1. Field of the Invention
The invention relates generally to antagonists of TGF-β activity, particularly to peptide antagonists of TGF-β activity. The invention also relates to methods of accelerating wound healing and preventing scarring by administering peptide antagonists of TGF-β activity to vertebrates.
2. Description of Related Art
Transforming growth factor β (TGF-β) is a family of 25-kDa structurally homologous dimeric proteins containing one interchain disulfide bond and four intrachain disulfide bonds. The TGF-β family is composed of three known members (TGF-β1, TGF-β2, and TGF-β3) in mammalian species. TGF-β is a bifunctional growth regulator: it is a growth inhibitor for epithelial cells, endothelial cells, T-cells, and other cell types and a mitogen for mesenchymal cells. TGF-β also has other biological activities, including stimulation of collagen, fibronectin, and plasminogen activator inhibitor-1 (PAI-1) synthesis, stimulation of angiogenesis, and induction of differentiation in several cell lineages.
TGF-β has been implicated in the pathogenesis of various diseases such as cancer, macular degeneration, intimal hyperplasia following angioplasty, tissue fibrosis (which includes integument scar tissue formation, liver cirrhosis, kidney fibrosis, lung fibrosis, heart fibrosis and others) and glomerulonephritis. It is known in the art that TGF-β plays an important role in scarring of the skin or organ fibrosis, which occurs as a result of injury or other fibrogenic stimulus. TGF-β's role in wound healing and scarring revolves around its activity as an important regulator of the extracellular matrix stimulating fibroplasia and collagen deposition and inhibiting extracellular matrix degradation by up-regulating the syntheses of protease inhibitors (see Roberts, 1995; Roberts and Sporn, 1996; and O'Kane and Ferguson, 1997). Neutralizing antibodies to TGF-β have been used experimentally to reduce scarring of wounds, to prevent lung injury in adult respiratory distress syndrome (ARDS), and to block restenosis following angioplasty in animal models. These promising results warrant the development of TGF-β antagonists (inhibitor) that might be useful in inhibiting, ameliorating or reversing the effects of TGF-β and treating diseases. However, practical applications have been limited by the large molecular size of the antibodies with resulting instability and poor tissue penetration (O'Kane and Ferguson, ibid;, Shah et al., 1994; Shah et al., 1995).
TGF-β peptide antagonists that block TGF-β binding to cell surface receptors and inhibit TGF-β-induced growth and transcriptional activation are described in copending U.S. application Ser. No. 09/095,637 and Huang et al., J. Biol. Chem. 272:27155-27160 (1997). The effective concentrations (EC50) of these peptide antagonists, with amino acid sequences corresponding to the 41st to 65th of TGF-β1 and TGF-β2, range from ˜60 nM to 1 μM, depending on the targeted TGF-β isoform. In contrast to TGF-β neutralizing antibodies, the peptide antagonists are relatively stable, exert rapid inhibitory actions, and can be applied topically. These properties suggest that they are useful for treating hypertrophic scarring in cutaneous wounds.
3. Related Art Citations
Throughout the instant specification, numerical citations in parentheses are used to cite specific references. Those references appear below and are herein incorporated by reference. No admission to the status of these references as prior art are made.                1. Derynck, R., Jarrett, J. A., Chen, E. Y., Eaton, D. H., Bell, J. R., Assoian, R. K., Roberts, A. B., Sporn, M. B., and Goeddel, D. V. (1985) Nature 316, 701-705.        2. Laiho, M., Weis, F. M. B., and Massagué, J. (1990) J. Biol. Chem. 265:18518-18524.        3. Madison, L., Webb, N. R., Rose, T. M., Marquardt, H., Ikeda, T., Twardzik, D., Seyedin, S., and Purchio, A. F. (1988) DNA and Cell Biol. 7:18.        4. Schlunegger, M. P., and Grutter, M. G. (1992) Nature 353:430-434.        5. Hinck, A. P., Archer, S. J., Qian, S. W., Roberts, A. B., Sporn, M. B., Weatherbee, J. A., Tsang, M. L.-S., Lucas, R., Zhang, B.-L., Wenker, J., and Torchia, D. A. (1996) Biochem. 35:8517-8534.        6. Liu, Q., Huang, S. S., and Huang, J. S. (1997) J. Biol. Chem. 1997 272: 18891-18895.        7. O'Grady, P., Kuo, M.-D., Baldassare, J. J., Huang, S. S., and Huang, J. S. (1991) J. Biol. Chem. 288:8583-8589.        8. Roberts, A. B. (1995) Transforming growth factor-β: activity and efficacy in animal models of wound healing. Wound Rep. Reg. 3,408-418.        9. Roberts, A. B., and Sporn, M. B. (1996) Transforming growth factor-β. In: Clark, R. A. F., ed. The Molecular and Cellular Biology of Wound Repair, 2nd ed. New York, N.Y., Plenum Publishing Corp., 275-308.        10. O'Kane, S. and Ferguson, M. W. (1997) Transforming growth factor βs and wound healing. Internat. J. Biochem. Cell Biol. 29, 63-78.        11. Shah, M., Foreman, D. M., and Ferguson, M. W. J. (1994) Neutralising antibody to TGF-β1,2 reduces cutaneous scarring in adult rodents. J. Cell Sci. 107, 1137-1157.        12. Shah, M., Foreman, D. M., and Ferguson, M. W. J. (1995) Neutralization of TGF-β1 and TGF-β2 or exogenous addition of TGF-β3 to cutaneous rat wounds reduces scarring. J. Cell Sci. 108, 985-1002.        13. Huang, S. S., Liu, Q., Johnson, F. E., Konish, Y., and Huang, J. S. (1997) Transforming growth factor β peptide antagonists and their conversion to partial agonists. J. Biol. Chem. 272, 27155-27160.        14. Kaufman, t., Levin, M., and Hurwitz, D. J. (1984) The effect of topical hyperalimentation on wound healing rate and granulation tissue formation of experimental deep second degree burns in guinea pigs. Burns 10, 252-256.        15. Knabl, J. S., Bayer, G. S., Bauer, W. A., Schwendenwein, I., Dado, P. F., Kucher, C., Horvat, R., Turkof, E., Schossmann, B., and Meissl, G. (1999) Controlled partial skin thickness burns: an animal model for studies of burn wound progression. Burns 25, 229-235.        16. Kitamura, M., Shimizu, M., Ino, H., Okeie, K., Yamaguchi, M., Funjno, N., and Nakanishi, I. (2001) Collagen remodeling and cardiac dysfunction in patients with hypertrophic cardiomyopathy: the significance of type IV and VI collagens. Clin. Cardiol. 24, 325-329.        17. Winter, G. D. (1974) Histological aspects of burn wound healing. Burns 1, 191-196.        18. Mutoe, T. A., Pierce, G. F., Morishima, C., and Deuel, T. F. (1991) Growth factor-induced acceleration of tissue repair through direct and inductive activities in a rabbit dermal ulcer model. J. Clin. Invest. 87, 694-703.        19. Asheroft, G. S., Yang, X., Glick, A. B., Weinstein, M., Letterio, J. J., Mizel, D. E., Anzano, M., Greenwell-Wild, T., Wahl, S. M., Deng, C., and Roberts, A. B. (1999) Mice lacking Smad3 show accelerated wound healing and an impaired local inflammatory response. Nature Cell Biology 1,260-266.        20. Zambruno, G., Marchisio, P. C., Marconi, A., Vaschieri, C., Melchiori, A.; Giannetti, A., and DeLuca, M. (1995) Transforming growth factor-β modulates β1 and β5 integrin receptors and induces the de novo expression of the αvβ6 heterodimer in normal human keratinocytes: implications for wound healing. J. Cell Biol. 129, 853-865.        21. Xia, Y.-P., Zhao, Y., Marcus, J., Jimenez, P. A., Ruben, S. M., Moore, P. A., Khan, F., and Mustoe, T. A. (1999) Effects of keratinocyte growth factor-2 (KGF-2) on wound healing in an ischemia-impaired rabbit ear model and on scar formation. J. Pathol. 188, 431-438.        22. Liu, Q., Ling. T.-Y., Shieh, H.-S., Johnson, F. E., Huang, J. S., and Huang, S. S. (2001) Identification of the high affinity binding site in transforming growth factor-β involved in complex formation with α2-macroglobulin: Implications regarding the molecular mechanisms of complex formation between α2-macroglobulin and growth factors, cytokines and hormones. J. Biol. Chem. 276, 46212-46218.        